Modulating TCR signaling to boost antitumor immunity

Abstract

CD8 T cells can be pivotal in controlling tumor growth. CD8 T cell function requires TCR recognition of cognate peptide presented on Major Histocompatibility Complex I (MHC-I). Therefore, the tumor-associated peptide repertoire can dictate the extent of T cell activation and effector function. This poses challenges in cancer types with low mutational burdens since there are not many peptides available for CD8 T cell recognition. In those settings, it is primarily self-peptides that remain for CD8 T cell recognition. CD8 T cells recognizing self-peptides often do so with low-affinity because higher-affinity TCR clones recognizing self-peptides are deleted in the thymus. One way to overcome the lack of high-affinity TCR clones in the tumor is to amplify TCR signaling downstream of the low-affinity TCR. Therapeutic avenues that augment downstream TCR signaling to overcome lower-affinity contacts remain underexplored. Diacylglycerol kinases α/ζ (DGKs) are important kinases that convert diacylglycerol (DAG) to phosphatidic acid, thereby quenching DAG signaling. DAG signaling is necessary for robust T cell activation and effector functions because it activates the AP-1 and NFκB pathways. Here, we used a novel DGKα/ζ inhibitor to augment downstream TCR signaling, and it ultimately led to increased T cell activation and effector function. We utilized TRP1high and TRP1low CD8 T cells, which recognize a peptide fragment from tyrosinase-related protein-1 (Tyrp1) with different affinities. TRP1high and TRP1low CD8 T cells exhibited greater proliferation and cytokine production when activated in the presence of DGKi and limited amounts of peptide. We engineered a pancreatic cell line to express low amounts of Tyrp1 (C2VTrp1 cells) to examine T cell effector functions. Effector TRP1high and TRP1low CD8 T cells induced greater cytolysis of C2VTrp1 cells in the presence of DGKi. Moreover, CD8 T cell killing of tumor cells was dependent upon interferon γ (IFNγ) and tumor MHC-I expression. DGKi administered to mice with αPD-1 augmented TRP1high and TRP1low CD8 T cell control of both melanoma and pancreatic tumors. TRP1high and TRP1low CD8 T cells also produced greater quantities of IL-2, IFNγ, and TNFα in mice bearing pancreatic tumors. Collectively, we demonstrate that DGKα/ζ inhibition decreases the TCR affinity threshold to augment tumor control. IFNγ is instrumental in antitumor immunity. Here, we demonstrate that CD8 T cell killing of C2VTrp1 cells was mediated by IFNγ, but not perforin. However, the mechanism of IFNγ-mediated tumor cell killing remains to be determined. We demonstrate that one TCR clone, TRP1high CD8 T cells, can eliminate tumor cells in different ways. TRP1high execution of B16 tumor cells was perforin-dependent, but TRP1high did not require perforin to kill C2VTrp1 tumor cells. While IFNγ did not directly induce cell death in C2VTrp1 cells, tumor-intrinsic IFNγ signaling was important for CD8 T cell killing. Moreover, soluble factors that were produced during TRP1high coculture with C2VTrp1 tumor cells do not directly induce cell death. TRP1high CD8 T cells can elicit bystander killing of tumor cells lacking MHC-I if they engage their cognate antigen on nearby tumor cells. Here, we confidently demonstrate that IFNγ does not directly kill C2VTrp1 tumor cells. The mechanism directly responsible for cell death remains to be elucidated and is being actively investigated. Collectively, we demonstrate that therapeutically amplifying downstream TCR signaling can lead to increased CD8 T cell effector functions and improved tumor control.